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KDM5A suppresses PML-RARα target gene expression and APL differentiation through repressing H3K4me2

ABSTRACT: Epigenetic abnormalities are frequently involved in the initiation and progression of cancers including acute myeloid leukemia (AML). A subtype of AML, Acute promyelocytic leukemia (APL), is mainly driven by a specific oncogenic fusion event of PML-RARα. PML-RARα was reported as a transcription repressor through the interaction with NCoR/HDAC complexes leading to the mis-suppression of its target genes and differentiation blockage. While previous studies were mainly focused on the connection of histone acetylation, it is still largely unknown whether alternative epigenetics mechanisms are involved in APL progression. KDM5A is a demethylase of histone H3 lysine 4 di- and tri- methylations (H3K4me2/3) and a transcription corepressor. Here, we found that the loss of KDM5A led to APL NB4 cell differentiation and retarded growth. Mechanistically, through epigenomics and transcriptomics analyses, we detected KDM5A binding in 1,889 genes, with the majority of the binding events at promoter regions. KDM5A suppressed the expression of 621 genes, including 42 PML-RARα target genes primarily by controlling the H3K4me2 in the promoters and 5’ end intragenic regions. In addition, a recently reported pan-KDM5 inhibitor, CPI-455 on its own could phenocopy the differentiation effects as KDM5A loss in NB4 cells. CPI-455 treatment or KDM5A knockout could greatly sensitize NB4 cells to ATRA induced differentiation. Our findings indicated that KDM5A contributed to the differentiation blockage in the APL cell line NB4, and inhibition of KDM5A could greatly potentiate NB4 differentiation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE152397 | GEO | 2021/04/01

REPOSITORIES: GEO

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Project description:We defined the RARα interactome in the MDA-MB453 breast cancer cell line genetically engineered to over-express an N-terminally tagged version of the nuclear retinoic acid receptor. Twenty eight nuclear proteins which interact with RARα and whose interaction is stimulated or reduced by the pan-RAR ligand, all-trans retinoic acid (ATRA) were identified. Given the potential significance of the S100A3 calcium-binding protein in the control of tumor progression, we focused our attention on this factor. Using the two models represented by the ATRA-sensitive SKBR3 and MCF7 breast cancer cell lines characterized by constitutive expression of S100A3 and RARα, we demonstrate that the endogenous forms of S100A3 and RARα interact in physiological conditions. The interaction of S100A3 with RARα is cell context independent and it is observed not only in breast cancer but also in acute promyelocytioc leukemia (APL) cells, characterized by expression of the RARα-derived PML-RARα oncogene, which is the product of the t(15:17) chromosomal translocation. S100A3 interacts directly and specifically with RARα and PML-RARα, being unable to bind other members of the RAR/RXR family of retinoid nuclear receptors. The interaction surface maps to the carboxyl-terminal region of the RARα ligand binding domain. Binding of S100A3 to RARα and PML-RARα controls the constitutive and ATRA-dependent degradation of the two receptors. Silencing of the S100A3 gene decreases the amounts of RARα in breast SK-BR-3 and lung A549 cancer cells, rendering them more refractory to the anti-proliferative action of ATRA. In SK-BR-3 cells, this effect is accompanied by a decrease in the lactogenic/differentiating action of ATRA. In APL-derived NB4 cells, S100A3 knock-down reduces the amounts of both RARα and PML-RARα. Contemporaneous down-regulation of the two receptors is associated with an increase in the basal and ATRA-induced expression of many granulocytic differentiation markers. Opposite on RARα and PML-RARα levels as well as ATRA induced differentiation markers are observed upon over-expression of S100A3 in NB4 cells.

Project description:The aim of this study is to identify microRNAs (miRNAs) transcriptionnally regulated by retinoic acid (RA). For that purpose we have used the RA-based treatment of the Acute Promyelocytic Leukemia (APL) as a model. This malignancy is characterised by a differentiation arrest of granulopoiesis at the promyelocytic stage. APL is molecularly associated with reciprocal translocations that always involve the retinoic acid receptor a (RARa). In the vast majority of APL cases, a t(15;17) chromosomal translocation fuses the genes encoding the promyelocytic leukemia protein PML and RARa. The resulting PML-RARa is a transcriptional repressor that impedes the expression of RA-regulated genes notably through an aberrant recruitment of transcriptional repressors and histone deacetylases. Consequently, these genes become insensitive to physiological doses (nanoM) of all-trans-retinoic acid (ATRA) but pharmacological doses (microM), overcome the PML-RARa-mediated repression and restore normal transcription and granulocytic differentiation. The restorative effects of RA can be reproduced in vitro in the NB4 cells, which were derived from an APL patient. These cells provide an excellent model to study the transcriptional deregulations that arise in APL and the molecular effects of the anti-cancerous RA-based treatment. We also used in this study the RA-resistant cells, namely NB4-LR1 and NB4-LR2 cells. The NB4-LR1 cells do transcriptionally respond to ATRA but do not maturate. In contrast, the NB4-LR2 cells show a clear defect in RA signaling, as they harbor a truncated form of PML-RAR protein that is not sensitive to pharmacological doses of RA. First, we plan to characterize miRNAs-repressed by PML-RAR. We reasoned that if some miRNAs are repressed by this protein, then pharmacological doses of RA should abolish this repression and lead to an increase in the level of expression of the corresponding miRNAs. NB4, NB4-LR1 and NB4-LR2 cells will thus be treated for 16h with ATRA (1 microM), and miRNAs profiles will be compared. We anticipate that, the expression of a potential miRNA-repressed by PML-RAR should be up-regulated by ATRA in both NB4 and NB4-LR1 cells but remain unchanged in NB4-LR2 cells. This expression pattern should in fact be similar to those observed for known RA-regulated genes, such as RARb, a well characterized target of the RARa and PML-RAR proteins. Importantly, this experimental procedure was already validated with the identification of a miRNA repressed by PML-RAR (patent Lecellier et al. #WO 2006/048553). MiRNAs candidates obtained will then be validated by chromatin immunoprecipitation using anti-RARa and anti-PML antibodies, followed by luciferase assays in presence or absence of ATRA. Keywords: retinoic acid-mediated gene regulation To characterize miRNAs regulated by PML-RAR.

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KDM5A suppresses PML-RARα target gene expression and APL differentiation through repressing H3K4me2

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